The synergetic effects of 4-nonylphenol and polyethylene microplastics in Cyprinus carpio juveniles using blood biomarkers

Microplastics are widely distributed in aquatic ecosystems along with other chemical pollutants. Therefore, it is vital to study the health-hazardous effects of MPs in combination with 4-nonylphenol (4-NP), which is a highly abundant industrial waste and a critical alkylphenol endocrine disruptor. We investigated the effects of the exposure to polyethylene microplastics (PE-MPs), 4-NP, and their combination on blood biomarkers in Cyprinus carpio juveniles. Four study groups were treated for 15 consecutive days: (1) control group, (2) 10 mg/L PE-MP group, (3) 10 mg/L PE-MPs + 200 µg/L 4-NP group, and (4) 200 µg/L 4-NP group, followed by 15 days of recovery. Biochemical analyses showed that creatine kinase, lactate dehydrogenase, glucose, liver enzymes, total protein, and A/G ratios were significantly increased after exposure to PE-MPs, 4-NP, and the combination. Hematological parameters (RBC's, Hb, Ht, neutrophil percentage, and WBC's) were significantly decreased in the three exposure groups, whereas mean corpuscular volume and lymphocyte percentages were significantly increased. The 15-day recovery period improved most hematobiochemical parameters and PE-MP accumulation indices. Taken together, we demonstrated the hazardous effects of PE-MP and 4-NP combinations on C. carpio blood parameters and highlighted their potential risk to human health.

and Sayed and Soliman 41 . Fish were fed each day with commercial pellets at 3% of their body weight, and water changed every day (50%) and MPs-redosed in water (immersion method of exposure) every day to prevent waste accumulation. Study procedures terminated with six fish in each group numbed on ice to eliminate stress caused by processing 42 and fish was euthanized MS-222 (Millipore-Sigma-Aldrich, Oakville, ON, Canada; 0.5 g/L). After cutting the tail, blood collected in heparinized and non-heparinized tubes for hematological and biochemical assessments, respectively.
Hematological parameters. Blood samples (6/ group) were taken from the caudal vein into heparinzed tubes to measure Hematological parameters including counts comprising erythrocytes (RBC's), total white blood cells (WBC's), differential WBC's, hematocrit (Ht), hemoglobin concentrations (Hb) were performed using Auto Hematology Analyzer (Rayto RT-7600) according to Hamed et al. 39 and Hamed et al. 43 . Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated using the formulae mentioned by Dacie and Lewis 101 Biochemical parameters. Blood samples were taken from the caudal vein into non-heparinzed tubes to centrifugation at 5000 rpm for 5 min and then the serum was removed by subjecting the tubes, stored at − 20 °C until further analysis of the following blood parameters: albumin, globulin, total protein (TP), glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), After the recovery period, the hematological parameters showed no change, except for the following: the RBC, MCV, and MCH levels in the PE-MP group, MCHC levels and neutrophil percentages in the PE-MPs + 4-NP group, and monocyte percentages in the 4-NP group; all showed nonsignificant decreases. The MCV levels in the 4-NP group showed a nonsignificant increase. The Ht levels in the PE-MP group showed a significant decrease. The lymphocyte percentages in the PE-MP + 4-NP group showed a significant increase ( Table 1).
The effects of combinations on biochemical parameters. AST  www.nature.com/scientificreports/ After the recovery period, the biochemical parameters did not change, except for the following: TP and AST in the PE-MPs, 4-NP, and PE-MPs + 4-NP groups; LDH and glucose in the 4-NP-exposed group; and albumin in the PE-MP-exposed group showed nonsignificant increases.
Additionally, the A/G ratio in the 4-NP group and globulin in the PE-MP group showed a nonsignificant decrease. However, the A/G ratio in the PE-MPs + 4-NP group and albumin in the 4-NP group showed a significant decrease (P < 0.05) ( Table 2)

Discussion
MP contamination in aquatic environments is a growing hazardous health problem as aquatic animals eat and digest MPs. Living organisms may be affected by MPs increasing their bioavailability and uptake of sorbed co-contaminates of different types. The combined effects of MPs and sorbed co-contaminants in aquatic organisms remain to be fully verified 46 . Therefore, in this study, hemato-biochemical parameters were used to investigate the effects of C. carpio exposure to 10 mg/L PE-MPs, 200 µg/L 4-NP, and 10 mg/L PE-MPs + 200 µg/L 4-NP for 15 days and then 15 days of recovery.
To assess fish health, hematological parameters must be measured 47 . We observed considerable variations in hematological parameters between groups. Our results were similar to the data reported by Hamed et al. 39 and Hamed et al. 43 . Decreased RBC, Hb, Ht, and WBC levels with increased MCV levels and lymphocyte percentages were also reported by Hamed et al. 39 in Oreochromis niloticus exposed to MPs and by 43 in C. carpio exposed to PE-MPs. Additionally, in fish exposed to phosalone 48 , and copper oxide nanoparticles 49 . The catfish (Clarias gariepinus) was also affected by UVA exposure with respect to hematology and cell alterations 50 . Some of parameters with hydroxychloroquine in catfish (C. gariepinus), Sayed et al. 44 and Mekkawy et al. 51 , and Abou Khalil et al. 52 with African catfish (C. gariepinus) in the presence of 4-NP. The exposure of carp to PE-MPs, 4-NP, and PE-MPs + 4-NP caused anemia, which might have been attributed to hematopoietic tissue alterations 53 and 54 . This possibly occurred because of increased mechanical fragility in cell membranes, which we observed in our erythrocyte morphology 55 . Under laboratory conditions and after exposure to different pollutants, peripheral RBCs, Hb, and Ht were decreased 56 , which might have been due to the heme-dilution of blood which resulted from tissue damage 57 . The negative effects of NPs on lymphoid tissues in exposed fish may reduce total WBC counts 58 . The bioaccumulation of pollutants in tissues may decrease WBCs. As blood oxygen levels decrease, toxicity caused by plastics could be enhanced by decreased hemoglobin levels. Similar results were reported by Mukherjee and Sinha 59 as cadmium contamination response 39 as effect of MPs and 43 as an effect of PE-MPs. Damage to the immune system after MPs accordingly will cause damage to the defense system and animal health 60 .   41,56 . Most biochemical parameters were significantly increased after exposure to PE-MPs, 4-NP, and PE-MPs + 4-NP when compared with controls and were consistent with Hamed et al. 43 . The rise in CK, LDH, ALP, ALT, AST, TP, glucose with variation by increase and decrease in albumin and globulin observed in our study were similarly studied by Hamed et al. 39 with O. niloticus in the presence of MPs. Some parameters with hydroxychloroquine in catfish (C. gariepinus) 44 , 61 and 41 with African catfish in the presence of 4-NP, and 43 with common carp (C. carpio) in the presence of PE-MPs. Banaee et al. 62 and Nematdoost Haghi and Banaee 63 state that the higher levels of the enzymes (CK, AST, ALT, LDH, and ALP) in serum are regarded as biomarker for cell membranes damage and as a tool for diagnosing changes in the environment in ecotoxicological studies, these enzymes are indicative of lesions in the tissues. Furthermore, elevated glucose levels indicated that glycogen had disintegrated or that its absorption was restricted in the liver. By contrast, increased blood glucose levels may have been due to hepatic tissue glycogen disintegration or impaired glucose absorption 64 . Previously, damage to different organ membranes in fish was observed following increased enzyme activity (ALT, AST, and ALP) caused by paraquat and plastic particles 65 , and in Pomatoschistus microps, biochemical parameters were changed when exposed to MPs and pyrene or to MPs and/or nickel 66 . Body homeostasis is maintained by proteins that prevent fluid leakage throughout the body 60 .
Immune system diseases and other kidney and hepatic issues are assessed using TP, albumin, and globulin tests 67 . Stoyanova et al. 68 reported that the intensification of anaerobic metabolism could be measured by LDH activity due to environmental changes, pollution, and energy depletion. Changes in LDH, AST, ALT, CK, and ALP activities were shown to indicate tissue lesions and to reflect environmental changes in ecotoxicology 69 . ALT and AST levels were increased following hepatocyte damage (Komatsu et al., 2002) or impaired carbohydrate and protein metabolism 70 . Peralta et al. 71 and Ramos-Barron et al. 72 reported that increased albumin levels indicated hepatorenal tissue alterations. Wiegertjes et al. 73 demonstrated that increased globulin levels were viable immune responses. In Nile tilapia (O. niloticus) exposed to MPs, albumin, globulin, and TP levels were higher, potentially indicating a damaged liver 39 and in C. carpio induced by PE-MPs 43 . Osman et al. 50 showed that fish underwent hyperglycemia when exposed to UVA stress or heavy metals and other contaminants 74 .
High PE-MPs levels were observed in PE-MP-exposed fish when compared with controls. This may have been due to the entrance of plastic particles with the water flow to the fish's body. A significant concentration of MPs was reported in different zebrafish organs 75 . Additionally, significantly higher MPs were observed in O. niloticus after MP exposure for 15 days 39 . MP accumulation in zebrafish yolk sac and migration to other organs were observed during embryogenesis 76 . Furthermore, in mussels, MPs absorbed through the gut mucosa were The percentage of erythrocytes with morphological alterations and nuclear abnormalities, when exposed to treatments, was significantly increased when compared with controls. Poikilocytosis may be affected by several factors, e.g., increased RBC membrane fluidity, declined ATP levels, and inhibited membrane-bound enzymes 79 . Our results were similar to other fish pollutant studies: Hamed et al. 43 , who studied C. carpio exposed to PE-MPs; Sayed et al. 44 , who studied C. gariepinus exposed to hydroxychloroquine; Hamed et al. 80 , who studied the protective role of Spirulina platensis against cytotoxicity and genotoxicity induced by lead nitrates in C. gariepinus; Soliman et al. 49 , who studied the damage caused by copper sulfate and copper oxide nanoparticles in O. niloticus; and Sayed et al. 81 , who investigated Oryzias latipes exposed to 4-NP. Several studies reported that morphological and nuclear abnormalities in erythrocytes and MN were genotoxicity biomarkers following exposure to radiation and chemicals 51,[82][83][84][85][86][87][88][89] . These biomarkers are powerful assessment tools for genetic and cellular damage in eukaryotes as they reflect DNA damage, and are simple, reliable, and sensitive measurement tools 51,84,86,87,90 .
Our blood parameter data indicated that the damage caused by combined PE-MPs and 4-NP was higher than the damage caused by 4-NP alone, which may be due to MPs facilitating entrance of other contaminants into aquatic organisms 24,29,91 . Previous laboratory studies reported that MPs were ingested by aquatic organisms 77,92 . Besseling et al. 22 , Koelmans et al. 93 , and Chua et al. 94 reported that MPs may carry other contaminants, including plasticizers and POPs, such as polychlorinated biphenyls and polycyclic aromatic hydrocarbons 65,93,95,96 , and could facilitate interactions with metals, such as PE-MP-mediated silver uptake in Danio rerio 97 . Some ecotoxicological studies have provided evidence of metal ion adsorption by plastic containers 98,99 . HOCs strongly and chemically sorb onto MPs than natural sediments according to a study comparing sorption rates onto natural and manufactured particulates 30 . Oliveira et al. 65 reported that PE bead exposure significantly increased toxicant bioavailability in juveniles exposed to lethal pyrene concentrations.
After the 15-day recovery period, erythrocyte morphological alterations, nuclear abnormalities, PE-MPs, and hematobiochemical changes were apparent in all treated groups when compared with controls. Our results were supported by Martins and Guilhermino 100 , who reported that despite the depuration phase, MPs persisted in D. magna for many generations, whereas Hamed et al. 39 indicated that MPs were detected and generated hematobiochemical effects in Nile tilapia after the recovery period. Notably, after recovery, a faint improvement in some parameters was observed but was not similar to controls. A possible reason for this could be that a 15-day recovery was not enough for fish to eliminate the toxic effects of pollutants or the tissue has bo ability to restore their functions as normal after MPs-exposure 46 .

Conclusions
The synergistic effect of PE-MPs and 4-NP induced a high degree of increase in creatine kinase, lactate dehydrogenase, glucose, liver enzymes, total protein, and A/G ratios after exposure to PE-MPs, 4-NP, and the combination. Also, hematological parameters (RBC's, Hb, Ht, neutrophil percentage, and WBC's) were significantly decreased in the three exposure groups. The 15-day recovery period improved most hematobiochemical parameters and PE-MP accumulation indices. Hematological and biochemical issues in carp when compared with individual exposures, our data showed that the synergistic effect of PE-MP and 4-NP caused more serious damage than each single chemical in dose dependent manner.

Data availability
All data generated or analyzed during this study are included in the research article.